The introduction of foreign material (antigenic material) into the body of a vertebrate animal provokes an immune reaction, the intent of which is to prevent the antigenic material causing damage to the body and to facilitate the removal of such material from the body. The immune system achieves this by producing immunoglobulin molecules (hereafter referred to as antibodies) which have the property of selectively recognising and binding to characteristic sites on the antigenic material. These sites are known as determinants and an antigen may possess one or more such determinants. Antibodies generated by the immune system each have specificity to only one determinant but a number of different antibodies may be produced if the antigenic material against which antibodies are raised possesses more than one determinant.
The primary function of antibodies is to protect the body from harmful foreign material, by agglutinating it, thereby assisting the normal body processes to remove the material.
The agglutination of antigenic material by antibodies does however have a practical use outside the body in the field of blood grouping.
Red blood cells (erythrocytes) have on their surface a number of different, and distinctive antigenic determinants, the character of which determinants allows the classification of blood into groups, or types (for example, A, B, O, A.sub.1 B, A.sub.2 B, B.sub.cord). It is essential in the transfusion of blood from a donor to a recipient that the transfused blood be of the same group as that of the recipient's blood, for if it is not, the immune system of the recipient will generate antibodies against the unfamiliar determinants upon the surface of the transfused erythrocytes. The reaction of such antibodies with foreign erythrocytes forms the basis of the technique of blood grouping.
In broad terms the grouping of blood samples relies upon the detection of agglutination or otherwise of erythrocytes in the sample when antiserum to the determinants found on the surface of that group of erythrocytes is added. The agglutination is a macroscopic effect which can be readily discerned by eye or detected automatically by machine.
The major source of blood grouping reagents has hitherto been through the hyperimmunisation of human subjects. This involves the introduction into a human subject of a substantial, but non-lethal, dose of a blood serum of a type different from that of a human subject. This provokes the normal immunological response resulting in the production of antibodies in the blood of the subject, a sample of which blood may be subsequently removed and an antibody preparation made therefrom. Such a preparation may be used to group an unknown blood sample since it will cause a visible flocculation or agglutination of the erythrocytes if the unknown blood sample is of the same type as originally introduced into the human serum donor.
In practice there are two types of blood grouping test. (Dunsford, F.; Bowley, C.: Techniques in blood grouping: 2nd Ed (1967). In the first, a sample of blood to be grouped is placed upon a blood grouping tile (see: British Pharmacopeoia: Determination of ABO donors). This is then mixed upon the tile with a sample of antiserum. Any subsequent agglutination indicates that the unknown sample of blood being grouped belongs to the same blood group as the group against which the antiserum was raised. In practice such tile agglutination grouping tests are routinely carried out in emergencies.
In the second type of experiment the blood sample to be grouped is placed together with an antiserum in saline solution in a tube and allowed to stand for a standard period (2 hours). The presence of agglutination may then be estimated by the sedimentation that has occurred within the standard period. Under emergency conditions a centrifuge may be used to expedite the test.
The efficacy of a particular blood grouping reagent is judged by the speed with which it forms agglutinants and by the manner in which its ability as an agglutinin varies with concentration.
The former of these criteria is commonly referred to in the art as "avidity". The avidity time of a particular blood grouping reagent is defined as the time taken for the mixing of the blood sample with the reagent to the time at which a noticeable agglutination of the sample has occurred.
In order to determine the dilution characteristics of a blood grouping reagent a saline agglutination titre may be measured. This measurement comprises preparing a number of equal volume, 2 fold, series, saline dilutions of the blood grouping reagent to be tested. To each dilution sample is added a known amount of the appropriate erythocyte suspension. The same amount of suspension is added to all dilutions. Each dilution sample is left to stand for a standard period (usually 2 hours) at the end of which an agglutination count is made under a microscope. The sample of highest dilution at which substantial agglutination occurs is determined and that dilution is termed the "saline agglutination titre". A reagent having a high saline agglutination titre is therefore a potent agglutinin.
Two problems are evident with the production of antibodies to erythrocyte determinants using the technique of hyperimmunisation of a human subject. Firstly, donations of blood serum are in limited supply and nowadays with the increasing frequency of major surgery the need for blood grouping has increased markedly. This places a strain on the supply of human blood serum which is also in damand for other medical uses. In addition, the agglutination effect of naturally produced antibodies to erythrocytes tends to give somewhat variable results. One reason for this effect is that the immune response prepares a `cocktail` of antibodies each component of which cocktail has a specific action on a determinant as discussed above. It is impossible to separate the various antibodies in this cocktail and so conventional antisera contain mixtures of antibodies and the mixtures vary from animal to animal (even within the same genus and from day to day). The same set of determinants is not present on all erythrocytes of the same group, which results in the response being in many cases very variable.
In summary a reagent suitable for blood grouping must be:
(1) specific for the appropriate antigen,
(2) sufficiently potent to give good macroscopic reactions to the weaker blood groups by emergency as well as routine methods of blood grouping,
(3) stable under the conditions of use, and
(4) readily available at reasonable cost.
Recent advances in molecular biology have provided a technique for the production of highly specific antibodies by the production of a hybrid cell (or hybridoma) from an antibody-producing spleen cell and a myeloma cell. This technique, the work of Kohler and Milstein (Eur. J. Immunol. 6, 292-295 (1976); Nature 256, 495-497 (1975); Eur. J. Immunol. 6, 511-519 (1976) provides a method for producing a limitless source of antibody. The antibodies produced are termed monoclonal antibodies since the hybrid cell elaborating them produces only one type of immunoglobulin molecule i.e. only immunoglobulin specific to one determinant. Hybridoma cells combine the two desirable features of myeloma cells and lymphocytes. That is myeloma cells have an immortal character and can self-replicate in vitro, whilst lymphocytes have the desirable property of expressing antibodies. Such hybrid cells are therefore a permanent source of pure, defined immunoglobulin. The process used for producing hybrid cells commonly comprises the steps of immunising a mouse with the appropriate antigen and after allowing sufficient time for the immune reaction to take place, sacrificing the animal and removing its spleen. A cell suspension may then be prepared from the spleen, and this suspension is mixed with a suspension of mice myeloma cells. Polyethylene glycol may be used to promote fusion of the two cells. The resulting individual hybridoma cultures, having derived from one lymphocyte cell, specifically produce one type of antibody, that is an antibody specific to one particular determinant.
This technique has been used by Voak et al (Vox Sanguinis 39 134-140 (1980)) to produce a monoclonal antibody to the determinants of A-type erythrocytes and such monoclonal anti-A's have been shown to be useful blood grouping reagents.
However, it has widely been thought that the immunisation of mice wth B-type human blood cells will not produce spleen cells capable of fusion with myeloma cells to form hybridoma cells elaborating anti-B immunoglobulin.
We have surprisingly found that this is not the case and that successful fusions may be readily obtained, resulting in hybridoma cells expressing monoclonal anti-B with high efficiency thereby producing a high avidity specific blood grouping reagent with a useful saline agglutination titre profile. Furthermore, it has proven possible to define an equilibrium constant and a dissociation rate constant for the immunocomplex formed between the monoclonal anti-B and B type blood cells. Hitherto such quantitative analysis of the strength of the immunocomplex formed between a blood grouping reagent and red blood cells has not been possible since previously known blood grouping reagents have comprised mixtures of immunoglobulin of differing specificity. The best figure that could be achieved formerly was therefore an average value.